Purification,Characterization And Identification Of Polyphenol Oxidase And Its Mechanism Of Enzymatic Browning In Longan Pericarp

In this study,the pericarp of longan（Dimocarpus longan Lour.cv.‘Fuyan’）was used as material,the polyphenol oxidase（PPO）isomers were extracted and purified.The sequences of the tryptic peptide fragments digested from PPO were analyzed by MALDI tandem mass spectrometry and then,the c DNA encoding the PPO was cloned subsequently.The transcriptional changes of PPO gene in pericarp during fruit development and postharvest storage were determined by fluorescence quantitative PCR.The characteristics and catalytic mechanism of the PPO isomers were compared and analyzed.The fluctuations of the PPO activity and the main polyphenolic substances in longan pericarp during fruit growth and storage were investigated.The main results were as follows:1.Isolation and purification of PPO from longan pericarpThe PPO was extracted from mature longan pericarp and,Four main PPO isomers named A1,B1,B2 and B3,were purified through column chromatography of Streamline Phenyl,DE-52,Macro-prep Q,Phenyl Sepharose HP and Sephacryl S-200,respectively.The results of SDS-PAGE and gel staining showed that,the purified PPOs in longan pericarp are of glycosylation-modified protein,and the apparent molecular weights of A1,B1,B2 and B3 were estimated to be 116.8 k Da,116.8 k Da,109.4 k Da and 102.5 k Da,respectively.2.Identification and c DNA cloning of PPO in longan pericarpThe purified proteins were conducted to SDS-PAGE and trypsin digestion and,an identical peptide fragment（m/z 1240.4）among them was demonstrated,and its sequence was identified as AVHYYDFVVK by MALDI-TOF MS/MS analysis and Macot database search,suggesting that the four isomers of PPO are all encoded by a same c DNA highly homologous to that of laccase.The Dl＿laccase c DNA was cloned,and the complete sequence was obtained by 3′-RACE and 5′-RACE.The results of analysis revealed that,the c DNA contained an open reading frame of 1701 bp in lengh,encoding a polypeptide of 566 amino acid residues;the polypeptide coded by the c DNA contained a puptative23-mer signal peptide on the N-terminus.3.Characterization of the PPO isomers in longan pericarpThe optimum temperature of A1,B1,B2 and B3 were 40°C,20°C,50°C and 40°C,respecively.When the temperature was lower than50°C,the enzyme activity of the four isomers remained stable.The optimal p H for A1 and B2 was 7.0,and the optimal p H for B1 and B3was 7.5,respectively.The results of substrate specificity analysis showed that,the PPOisomers from longan pericarp were quite different from that of the laccases from other species.The common polyphenolic chemicals,such as catechol,4-methylcatechol,resorcinol,gallic acid,pyrogallic acid and chlorogenic acid,did not serve as substrates.The PPO isomers revealed high affinity for（-）-epicatechin（EC）,and poor affinity for（（10））-catechin（C）and（-）-epicatechin gallate（ECG）,indicating that the PPO in longan is an EC oxidase with high substrate specificity.The Km values of the A1,B1,B2 and B3 isomers for EC were 0.159 mmol/L,5.0 mmol/L,0.111mmol/L and 0.225 mmol/L,respectively.High concentration of metal ions Fe³⁺,Cu²⁺and Mn²⁺had certaineffects on the enzyme activities of the isomers,while CTAB,DTT,L-cysteine and ascorbic acid had obvious inhibitory effects on EC oxidation catalyzed by the PPO.4.UPLC-MS analysis of the oxidation products of EC catalyzed byPPOThe results revealed that,the PPO from longan pericarp mainlycatalyzed the condensation of EC.Different types of oligomer were detected in the enzymatic products of EC,and the generation time of the different oligomers was different.Dimers were mainly produced in the reaction time of 1?3 min,and trimers and tetramers were mainly in 3?5min and 5?10 min,respectively.The results of UPLC-MS analysis also showed that the PPO could not catalyze the oxidation of phenolic substrates such as C,ECG,gallic acid and chlorogenic acid,which was consistent with the results of spectrophotometric determination.However,in the presence of EC,the PPO could catalyze the degradation of these substances.The hetero-dimers of EC and gallic acid,chrologenic acid,C and rutin were detected,respectively in the PPO-EC-polyphenolic reaction system,suggesting that,the oxidation product of EC catalyzed by logan PPO can condense with other polyphenolics non-enzymatically.Corilagin and gallic acid inhibit significantly the oxidation of EC suggesting their inhibitory effects on the activity of PPO,while ascorbic acid inhibits the polymerization of EC and the formation of oligomer.5.Changes of PPO activity and Dl＿laccase expression in longanpericarp during fruit growth and postharvest storageThe PPO activity of longan pericarp reached its peak at 40 days after anthesis during the fruit growth and development.Under the storage condition of 25°C,the activity of PPO increased firstly and then decreased;while under low-temperature storage at 3±1°C,its activity decreased gradually.The results of q RT-PCR demonstrated that,during the development and maturation of fruilt,an expression peak of Dl＿laccase was detected at50 days after anthesis.During the postharvest storage,the transcription level increased at 2 days after storage at 25°C,and 7 days after storage at4°C.6.UPLC-MS determination of polyphenolics in longan pericarpThe results showed that longan pericarp mainly contained ellagic acid,corilagin,chorogenic acid,EC,procyanidin B2,rutin and other phenolic substances.During the growth and development process of longan fruit,the phenolic compounds contents in the pericarp increased firstly and then decreased.The phenolic compounds decreased significantly with the process of fruit postharvest senecense and pericarp browning.